Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment

Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment Q2

Ewa Taracha a, *, Ewelina Kaniuga a, Stanis1aw J. Chrapusta b, Piotr Maciejak a, c, d, e  , Adam Hamed a, Pawe1 Krza˛ scik c Lech Sliwa a

Department of Neurochemistry, Institute of Psychiatry and Neurology, 9 Sobieskiego St., 02-957 Warsaw, Poland  skiego St., 02-106 Warsaw, Poland Department of Experimental Pharmacology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawin Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, 26/28 Krakowskie Przedmiescie St., 00-927 Warsaw, Poland d Institute of Physiology and Pathology of Hearing, 10 Mochnackiego St., 02-042 Warsaw, Poland e World Hearing Centre, 17 Mokra St., Kajetany, 05-830 Nadarzyn, Poland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 January 2014 Received in revised form 7 April 2014 Accepted 9 April 2014

Behavioral sensitization and tolerance to repetitive exposure to addictive drugs are commonly used for the assessment of the early stages of the drug dependence progress in animals. The orchestra of tools for studying the progress of drug dependence in laboratory rodents has been considerably enriched in the 1980s by the introduction of ultrasonic vocalization (USV) detection and characterization. However, the relationship between the results of this technology and those of traditional behavioral tests is not clear. We attempted to elucidate some of the respective ambiguities by comparing the effects of an intermittent amphetamine treatment, which was aimed both at the induction of sensitization and tolerance to this drug and at testing the persistence of these effects, on the locomotor activity and 50-kHz USV responses to both the drug and the context of drug exposure in adult male rats showing diverging susceptibility for sensitization to amphetamine. Categorization of the rats into low and high responders/ callers based on sensitization of their frequency-modulated 50-kHz USV responsiveness showed some correspondence with conditioned place preference effects, but not with responses to amphetamine. The study showed distinct changes in the rate and latency of the frequency-modulated 50-kHz USV responses to repetitive amphetamine treatment, which were reminiscent of classical behavioral signs of sensitization and tolerance. These results show the utility of the appetitive USV for monitoring of early phases of complex processes leading to drug dependence. However, USV, locomotor activity and conditioned place preference seem to reflect different aspects of these phenomena. Ó 2014 Published by Elsevier Ltd.

Keywords: Drug dependence Individual differences Place preference Sensitization Tolerance Ultrasonic vocalization

1. Introduction The ability to stimulate brain reward system(s) and to produce long-lasting behavioral sensitization and tolerance are typical properties of addictive drugs, which are utilized for modeling, in laboratory animals, early phases of complex processes leading to

Abbreviations: Amph, amphetamine; ANOVA, analysis of variance; CPP, conditioned place preference; DAMGO, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin; FM, frequency-modulated; HC, high callers; LA, locomotor activity; LC, low callers; Sal, physiological saline; TIPS, two-injection protocol of sensitization; USV, ultrasonic vocalization. * Corresponding author. Tel.: þ48 22 458 2619; fax: þ48 22 458 2771. E-mail address: [email protected] (E. Taracha).

drug addiction. For many years these characteristics were assessed but indirectly, using, inter alia, locomotor activity- (LA) -related and conditioned place preference (CPP) tests. Since 1980s, a novel approach employing detection and measurement of ultrasonic vocalization (USV) is being developed for animal studies on behavioral effects of drugs of abuse. This technique allows one to assess motivational and emotional states of laboratory rodents by exploiting a variety of USV features (Brudzynski, 2013; Knutson et al., 2002; Wang et al., 2008; Wöhr et al., 2009; Wöhr and Schwarting, 2009). With regard to the assessment of drugs of abuse-induced positive affective states, the most useful is the socalled appetitive 50-kHz USV (Brudzynski, 2009; Burgdorf et al., 2009; Ma et al., 2010; Mahler et al., 2013; Maier et al., 2012; Mu et al., 2009; Simola et al., 2012, 2014; Thompson et al., 2006;

http://dx.doi.org/10.1016/j.neuropharm.2014.04.008 0028-3908/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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Wright et al., 2010, 2012), which is considered a substitute of drug addicts’ self-reporting (Barker et al., in press; Panksepp and Burgdorf, 2000; Mahler et al., 2013). Fifty-kHz USV reaction to drugs of abuse can picture their rewarding effect and its sensitization as well as the reaction to the context of drug exposure (Barker et al., in press; Hamed et al., 2012; Maier et al., 2012; Taracha et al., 2012). Experimental data evidence a convergence between subjective perception of the psychoactive action of these substances and CPP (Ahrens et al., 2013; Burgdorf et al., 2007), and a positive correlation between the rate of 50kHz USV and the acquired self-administration of such drugs (Browning et al., 2011; Maier et al., 2010). Our recent study, the results of which have been confirmed in general by the report of Ahrens et al. (2013), has shown that the positive affective staterelated FM 50-kHz USV response to amphetamine is greatly diversified among nonselected rats, but shows a substantial intraindividual stability: the rats that experienced a strong sensitization from the first drug dose showed persistence of this effect after consecutive doses, while those resistant to the sensitization did not sensitize during the further drug treatment (Taracha et al., 2012). Diverging vulnerability to drug dependence/addiction, which is well documented by both animal and human studies (Cain et al., 2005; Pelloux et al., 2006; Zuckerman, 1984), is known to correlate negatively with the results of anti-addiction therapies. Our recent results (Taracha et al., 2012) opened a perspective for the creation of a rat model that includes this characteristic, and this study represents the next step in the development of such a model. In particular, we intended to verify the validity of our previous categorization of the susceptibility for drug dependence (based on sensitization of FM 50-kHz USV response to amphetamine) by confronting it with the results of classical behavioral tests of proven utility in this regard (CPP and LA), to refine our categorization by incorporating a FM 50-kHz USV and LA response-based assessment of drug tolerance, and possibly to seek an interpretation of the USVrelated findings in their comparison with CPP and LA data. 2. Methods and materials 2.1. Subjects Thirty-six male SpragueeDawley rats from the stock of the Mossakowski Medical Research Centre, PAS, Warsaw, Poland, were used for the study. The rats were 10e11 weeks old upon arrival and were housed six per opaque plastic cage (55  33 cm floor size, H ¼ 19.5 cm) in a temperature- and humidity-controlled room (21  2  C, 60e70% relative humidity) under a 12-h light/12-h dark cycle (lights on at 7 a.m.), with free access to standard laboratory rat chow and tap water. The rats were randomly divided between the vehicle-treated subset (Ctrl, N ¼ 6) and the amphetamine-treated subset (N ¼ 30); there was no significant difference in body weight between these subsets (mean  SD: 305  9 g and 288  29 g, respectively, p ¼ 0.18). 2.2. Habituation Before the start of USV/LA tests/recordings, the rats were acclimated for 1 week in the local animal facility. During this period, they were given six ‘daily’ (excepting weekends) sessions of graded habituation to the procedures related to drug treatment: 2 sessions of simple gentle stroking when on the experimenter’s hands (for about 1 min), followed by 2 sessions of being hand-immobilized exactly as for ip amphetamine injections, followed by 2 sessions of being hand-immobilized and pricked with an injection needle of the size intended for ip injections. After each session, the rats from a given cage were placed in a cage identical with the housing cage, with some clean wooden chips on the bottom, and then returned to their home cage. The post-handling cage was replaced with a fresh one for each group of cagemate rats. 2.3. Experimental design Amphetamine was given to rats in a way that allows to follow-up the development and to verify the stability of sensitization and tolerance to repeated drug exposures. The procedure involved: 1) the initiation and verification of sensitization to the drug with the so-called two-injection protocol of sensitization (TIPS) that consisted of administration, at 6-day interval, of two drug doses (Amph1 and Amph2); this protocol has been first used for the induction and assessment of

locomotor sensitization (with no interfering tolerance) of mice to cocaine and morphine (Valjent et al., 2010), and was next successfully employed (Taracha et al., 2012) for the induction and verification of sensitization of LA and 50-kHz USV responses to amphetamine in rats; 2) the development of tolerance with continuing intermittent drug treatment (Amph3-Amph9); since frequent dosing facilitates the development of tolerance, these doses were given at 1-day intervals, excepting the weekend-related breaks; and 3) the verification of the persistence of these effects with amphetamine challenge (Amph10) given after 2-week withdrawal from the treatment. A complete scheme of the experimental design is shown in Fig. 1. All efforts were taken to minimize animal suffering and the number of rats used. All animal use procedures were in accordance both with the European Communities Council Directive of November 24, 1986, on the protection of laboratory animals (86/ 609/EEC), and with the current laws of Poland, and were approved by the Bioethical Committee of the Medical University of Warsaw (Certificate of approval No. 47/ 2012).

2.4. Drugs D-Amphetamine sulfate (Sigma, St. Louis, MO, USA) was dissolved (1.5 mg/ml) in  ski, Poland) and sterile aqueous 0.9% NaCl solution (Sal; Polpharma, Starogard Gdan injected ip at the dose of 1.5 mg/kg body weight. As a rule, the preferred compartment of CPP apparatus (see below) was used for Sal injections and post-Sal USV/LA recording sessions, and the non-preferred section was used for amphetamine injections and post-drug USV/LA recording sessions. However, the sessions preceding Amph2, Amph9 and Amph10 injections were carried out with the entire CPP apparatus open for the subject rats.

2.5. CPP apparatus and test procedure The apparatus consisted of a plywood box (with 34 cm high walls) divided into two main compartments (35.5  20 cm floor size) separated by a smaller section (10  20 cm floor size) with vertically sliding matt black doors and floor and walls coated with clear lacquer. One main compartment had a rough plywood floor and both its floor and the walls were painted matt black. The other main compartment had a smooth black plastic floor and its walls were also painted matt black, but the lowermost part of its long walls carried four 4 cm high white paint rectangles of 10, 5.5, 5.5 and 8 cm length (listed from the most proximal to the most distant with regard to the door), spaced 2 cm one from another. The CPP test was performed in a 4.4  2.8  2.9 m (L  W  H) room with ceiling and walls painted white and lit with four reflectors (each facing different wall) fixed centrally 48 cm below the ceiling, each equipped with an incandescent 40 W matt white light bulb. Two CPP apparatuses were used simultaneously, which were thoroughly cleaned after each rat. The apparatuses were separated with a 1.1  0.7 m  (L  H) sound-attenuating wall made of a 2 cm thick particle board with black veneer on both sides; the wall has been verified to prevent the microphones used for USV recording from collecting calls emitted by the rat residing in the CPP apparatus behind the wall. Rats’ LA was registered with a ceiling-fixed model EV-650CG video camera (Sony, Japan) connected to a PC equipped with the EthoVisionÒ XT Video Tracking System v.7 (Noldus Information Technology B.V., Wageningen, The Netherlands). Initial place preference was determined in a 15-min pre-test, with no USV and LA recording. On the next day, the rats were given an ip Sal injection and were instantly confined to the preferred section of the apparatus for a 40-min session of LA and USV recording. One day later, the Ctrl rats and the rats scheduled for drug treatment were given their next Sal dose and the first amphetamine dose, respectively, and were immediately confined to the non-preferred section of the apparatus for another 40-min session of LA/USV recording. Seven days after the initial Sal injection, all rats were again given access to open CPP apparatus for 20 min (of which the first 15 min were taken for CPP analysis) for the assessment of their place preference and LA/USV activity. CPP was calculated as the difference between the times spent in the drug-paired section during the test and pre-test.

2.6. USV recording USVs were recorded with a single CM16 condenser microphone (Avisoft Bioacoustics, Germany) placed 35 cm above the cage/testing box floor, centrally with regard to the rat-accessible area. The microphone was sensitive to frequencies of 15e180 kHz, had a flat response characteristic (6 dB) within the 25e140 kHz frequency range, and was connected to a custom-made amplifier of 600 U input impedance, 16 V/V (12 dB) voltage gain, and 0.1 dB (30 Hze100 kHz) frequency response. The amplified signals were passed to an adjacent room, processed with a custom-made antialiasing filter, and then sent to a PC equipped with PCI-703-16A acquisition board (14-bit, 400 kHz; Eagle Technology, Eagle River, WI, USA) and a custom-written software (Rat-Rec Pro 5.0), processed using a fast Fourier-transform (1024 or 512, Hamming or Hann window) and displayed as a color spectrogram. Frequency-modulated (FM) and non-FM (“flats”) 50 kHz calls were identified as specified elsewhere (Brudzynski, 2013). Since the number of flats is not affected by amphetamine treatment (Ahrens et al., 2009; also confirmed in our lab), only FM 50 kHz calls were analyzed.

Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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Fig. 1. Scheme of the experimental design with consideration of the selected phenomena associated with early phases of drug dependence progress. Please note that the labels under the time axis show the timing of experimental manipulations aimed at modeling of these phenomena and may not correspond with the actual duration of the underlying neurobiological processes.

2.7. USV-based rat categorization The rats with high and low susceptibility for FM 50 kHz USV sensitization to amphetamine were identified based on the difference (D) in the rate (RUSV, calls/ 20 min) of their FM 50-kHz USV responses to Amph2 (USV2) and Amph1 (USV1) in the TIPS paradigm:

DRUSV2USV1 ¼ RUSV2  RUSV1 as the measure of sensitization. The subset comprising the rats with DRUSV2USV1 of >2 S.E.M. below the mean for the entire amphetamine-treated cohort was termed ‘low callers’ (LC, N ¼ 12), whereas the subset that comprised the rats with an increase of >2 S.E.M. above the mean was termed ‘high-callers’ (HC, N ¼ 8). The remaining 10 rats were excluded from further statistical analyses. The HC group included a single rat in which aversive (22 kHz) USVs has been recorded during this study, which occurred 6e9 min after the injection of Amph2. Since no more aversive USVs was observed in this rat for the rest of the study, that episode was knowingly ignored and all other data for the rat were included in statistical analyses.

2.8. Statistical analysis Data are expressed as means  S.E.M. All data were first checked for homogeneity of variances using the BrowneForsythe test. The test revealed significant variance heterogeneity of FM 50-kHz USV rate and latency data, hence these data were square root-transformed prior to statistical analyses. CPP test-related time and USV data were analyzed by a 1-way ANOVA with rat group as the main factor. Except when specified otherwise, FM 50-kHz USV rate/intensity (numbers of calls/time unit), latency time and LA data were analyzed by a 2-way repeated measures ANOVA, with rat group (LC, HC, and occasionally Ctrl rats) as the main factor and drug dose (Amph1, Amph2, Amph9 and Amph10) and (in some cases) post-drug session time as the repeated measures factors. Because of transient equipment failure, the dataset for LC drug context-related USV rate and latency comprised data for 10 rats only. The significance of within-group and between-group differences was tested, when appropriate, by Student’s t-tests for dependent and independent variables, respectively, or by the Tukey test (as specified in the respective figure legend). To keep the family-wise probability of type I error <0.05, the sequential BonferronieHolm correction was applied to the results of the t-tests when appropriate. Spearman’s rank correlation coefficient (R) was used as a measure of association between variables. In all cases, a p < 0.05 was considered significant. All statistical analyses were performed using the Statistica v. 7.1 software package (StatSoft Inc., Tulsa, OK, USA) except for the BonferronieHolm correction that was applied ‘manually’.

FM 50-kHz USV rate in naïve HC and LC rats given Sal injection (CPP training, saline treatment session) was very low and did not differ between these groups (data not shown). The differences in TIPS-related changes in the rate of FM 50-kHz USV response between the entire cohort of drug-treated (unselected) rats and LC and HC rat subsets are shown in Fig. 2. Notably, the two subsets markedly differed also in the time course of the changes in their FM 50-kHz USV responses to amphetamine induced by their first exposure to the drug. Treatment of LC and HC rats with 7 additional ‘daily’ (5 doses a week) drug doses (Amph3eAmph9) followed by 2-week withdrawal and the final drug challenge (Amph10) resulted in further changes in the rate of FM 50-kHz USV response (Fig. 3, upper panel). Two-way ANOVA of the FM 50 kHz USV rate response to Amph, with rat group (LC, HC) as the main factor and drug dose (Amph1, Amph2, Amph9, Amph10) as the repeated measures factor, yielded significant effects of group, dose, and group  dose interaction, evidencing dissimilar changes in the USV response in these groups. Post-hoc analysis showed significantly stronger FM 50-kHz USV responses to Amph1, Amph2 and Amph9 in HC rats compared to LC rats. The daily drug treatment did not significantly affect the USV response in LC rats, but markedly weakened it in HC rats (Fig. 3, upper panel), indicating the emergence of tolerance. Similarly to the assessment of sensitization (see Section 2.7), the tolerance was assessed as the difference (D) in the rate of FM 50kHz USV responses (RUSV) to Amph9 and Amph2:

DRUSV9USV2 ¼ RUSV9  RUSV2 :

3. Results 3.1. Effects of TIPS and the ensuing amphetamine treatment on post-drug FM 50-kHz USVs Preliminary 2-way repeated measures ANOVA of the data for FM 50 kHz USV response to amphetamine for the entire drug-treated rat cohort (N ¼ 30; data not shown), with drug dose (Amph1 and Amph2), and post-drug time (ten 2-min intervals) as the repeated measures factors, yielded significant effects of the dose (F1,29 ¼ 63.0, p < 0.001), post-drug time (F9,261 ¼ 38.0, p < 0.001), and dose  post-drug time interaction (F9,261 ¼ 11.4, p < 0.001), indicating a major change in FM 50-kHz USV response to amphetamine induced by Amph1.

Fig. 2. Post-amphetamine session time-related representation of TIPS-evoked changes in the rate of FM 50-kHz USV response in nonselected rats and rat subsets categorized as low callers (LC) and high callers (HC). Two-way ANOVA results for LC and HC rats: group effect: F1,18 ¼ 57.4, p < 0.001, post-amphetamine time effect: F9,162 ¼ 17.9, p < 0.001, group  post-amphetamine time interaction effect: F9,162 ¼ 18.1, p < 0.001.

Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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developed over the period of daily amphetamine treatment (Amph2eAmph9) were significantly stronger in HC compared to LC rats (Fig. 4). 3.2. Effects of TIPS and the ensuing amphetamine treatment on post-amphetamine LA Amphetamine markedly stimulated LA in both LC and HC rats and there was a tendency for enhancement of this response with ensuing drug treatment, with no tangible between-group difference. Preliminary 2-way repeated measures ANOVA of LA response data for Amph1 and Amph2 yielded a significant effect of dose (F1,18 ¼ 11.1, p ¼ 0.004), but not of group (F1,18 ¼ 0.037, p ¼ 0.85), or of group  dose interaction (F1,18 ¼ 0.051, p ¼ 0.82). Two-way ANOVA of the full set of LA response data (including the data for naive rats’ response to Sal) showed qualitatively similar results. Post-hoc analysis revealed a significant, but slight, enhancement of this response over the course of daily drug treatment in HC rats, but this effect was lost after 2-week withdrawal period (Fig. 5). 3.3. Effects of repetitive amphetamine treatment on “anticipatory” FM 50 kHz USVs and LA

Fig. 3. Effect of repetitive amphetamine treatment on FM 50-kHz USV rate response (upper panel) and latency of the response (lower panel) in LC and HC rats. USV rate data ANOVA results: group effect: F1,18 ¼ 48.9, p < 0.001, dose effect: F3,54 ¼ 51.0, p < 0.001, group  dose interaction effect: F3,54 ¼ 8.83, p < 0.001. FM 50-kHz USV latency data ANOVA results: group effect: F1,16 ¼ 27.2, p < 0.001, dose effect: F3,48 ¼ 80.3, p < 0.001, group  dose interaction effect: F3,48 ¼ 4.25, p ¼ 0.01. * e p < 0.05, ** e p < 0.01, *** e p < 0.001 vs. the respective Amph1 value; þ e p < 0.05, þþ e p < 0.01 vs. the respective Amph2 value; @ e p < 0.05 vs. the respective Amph9 value; # e p < 0.05, ### e p < 0.001 vs. the corresponding HC group value.

However, HC rats still vocalized after the last daily dose (Amph9) markedly more than they did after Amph1. The final drug dose given to LC and HC rats after 2-week withdrawal from the daily treatment revealed further significant and marked potentiation of the USV response in LC rats, and a reversal of the suppressing effect of daily drug treatment on the response, but no further enhancement, in HC rats. Still, the average FM 50-kHz USV rate during the post-Amph10 session was about 2.5 times higher in the latter. Increases in FM 50-kHz USV rate response to amphetamine were associated with significant reduction in the latency of the response. In both LC and HC rats, post-hoc analysis showed a major reduction of the latency of the FM 50-kHz USV response to Amph2 (by 74 and 82%, respectively). In HC rats, the latency showed minor and nonsignificant fluctuations with further drug treatment, whereas in their LC counterparts a further significant decrease in the latency was found at Amph10 challenge. As a result, the between-group difference in the latency lost significance at the end of the daily amphetamine treatment and was negligible at the end of the study period (Fig. 3, lower panel). Both the sensitization of the FM 50-kHz USV response in the TIPS paradigm, and the tolerance in the USV response that

Repeated drug treatment evoked distinct changes in anticipatory FM 50 kHz USV. Two-way ANOVA gave significant effects of group, dose and group  dose interaction on both the rate and latency of anticipatory USVs. While there was no significant change in anticipatory USV rate in LC rats over the course of the study, this rate showed large variations in HC rats, which roughly paralleled the changes in the corresponding post-amphetamine FM 50-kHz USV rate. HC rats vocalized significantly and markedly more than LC rats prior to Amph2 injection. This difference greatly diminished over the period of daily drug treatment and lost significance at the end of the treatment (Amph9), mostly due to a drop in anticipatory USV in HC rats. After 2-week withdrawal from the treatment, the rate of anticipatory USV in HC rats rose again to match that found prior to the injection of Amph2. However, it did not differ significantly at this time point from the respective value for LC rats, due both to an increased inter-individual variability in the anticipatory USV and to some increase in anticipatory USV rate in LC rats (Fig. 6, top panel). There was no significant change in the latency of the anticipatory USV in either LC or HC rats during the course of amphetamine treatment. Still, the HC rats compared to the LC rats showed significantly shorter latency of the USV when awaiting Amph2 or the post-withdrawal Amph10 challenge, but not at the

Fig. 4. Comparison of amphetamine-induced FM 50-kHz USV sensitization and tolerance effects in LC and HC rats. ** e p < 0.01, *** e p < 0.001 vs. the respective HC rats value; Student’s t-test for independent variables. Please note that the data shown are difference values calculated as described in Sections 2.7 and 3.1.

Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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susceptibility to drug dependence, and what relevant information could be derived from rat USV responses to repetitive drug treatment. We sought answers to these questions by first verifying our USV-based categorization with the results of the respective CPP and LA tests, and next by comparing changes in FM 50-kHz USV and LA responses to amphetamine evoked by repetitive (daily) drug treatment and its discontinuation. 4.2. Repetitive amphetamine treatment effects: USV response Fig. 5. Effect of repetitive amphetamine treatment on post-amphetamine LA of LC and HC rats. ANOVA results (including the data on LA response to Sal in naive rats): group effect: F1,18 ¼ 0.28, p ¼ 0.60, dose effect: F4,72 ¼ 66.7, p < 0.001, group  dose interaction effect: F4,72 ¼ 0.82, p ¼ 0.52. ** e p < 0.01, *** e p < 0.001 vs. the respective Sal value; þ e p < 0.05 vs. the respective Amph1 value.

Compared to LC rats, HC rats showed much stronger FM 50-kHz USV responses at all stages of drug treatment. However, the withdrawal from daily amphetamine treatment resulted in further

end of daily amphetamine treatment (Fig. 6, middle panel). In contrast, there was no tangible between-group difference or within-group change in ‘anticipatory’ LA (Fig. 6, bottom panel). 3.4. CPP test and FM 50-kHz USVs during the test ANOVA of the CPP test data yielded significant differences between LC, HC and Ctrl rats in regard to the change in time spent in the drug-paired compartment. There was no difference between these groups (p ¼ 0.89) in regard to the results of the pre-test (not shown). Post-hoc test revealed that both the Ctrl and LC rats spent notably less time than HC rats in the drug-paired section (Fig. 7, top panel). ANOVA showed also significant difference(s) between these groups in FM 50-kHz USV rate, and the Tukey test evidenced significantly higher USV rate in HC rats compared to either Ctrl or LC rats (Fig. 7, middle panel). For all these subsets combined (N ¼ 26), there was no significant correlation between the time spent in the drug-paired section and FM 50-kHz USV rate or LA (R ¼ 0.28, p ¼ 0.16, and R ¼ 0.18, p ¼ 0.36, respectively). Over the course of the test, HC rats spent more and more time in the drugpaired section, but emitted less and less FM 50-kHz USVs (Fig. 7, bottom panel). 4. Discussion 4.1. Rat categorization The choice of criterion is of key importance in the studies on the diversity of individual’s reaction to a given stimulus. Some authors studying diversification of rat USV responses to addictive drugs categorize their cohorts based on the 50-kHz USV reaction to the first drug dose (Ahrens et al., 2013; Burgdorf et al., 2007). This approach capitalizes on rewarding properties of the drugs and is based on the assumption that the pleasing effect of the first dose will induce the subject to seek next exposures to the drug. In contrast, we categorized our rats based on the sensitization of their USV response, i.e. on a comparison of the response to the second vs. the first drug dose. We think this is a better way as it implicitly takes into account the actual while yet poorly understood neurobiological changes associated with the progress toward drug dependence. The two categorizations, however, may produce converging results, as in our study Amph1 induced significantly less FM 50-kHz USVs in LC than in HC rats. The diversity of individual USV responses may result both from the fact that the same stimulus can be perceived with different ‘intensity’ by different individuals, and from innate differences in their ability for the expression of the response. One might ask whether the inter-individual differences in FM 50-kHz USV response to addictive drugs reflect the diversification of individual

Fig. 6. Effect of repeated exposures of LC and HC rats to amphetamine on the rate (top panel) and latency time (middle panel) of their FM 50-kHz USV and on LA (bottom panel) during the 20-min USV/LA recording sessions preceding the injection of consecutive drug doses. USV rate data ANOVA results: group effect: F1,16 ¼ 9.41, p ¼ 0.007, dose effect: F2,32 ¼ 7.12, p ¼ 0.003, group  dose interaction effect: F2.32 ¼ 6.28, p ¼ 0.005. USV latency data ANOVA results: group effect: F1,16 ¼ 16.9, p < 0.001, dose effect: F2,32 ¼ 3.80, p ¼ 0.033, group  dose interaction effect: F2.32 ¼ 4.15, p ¼ 0.025. LA data ANOVA results: group effect: F1,18 ¼ 0.016, p ¼ 0.90, dose effect: F2,36 ¼ 1.25, p ¼ 0.30, group  dose interaction effect: F2.36 ¼ 0.62, p ¼ 0.54. * e p < 0.05 vs. the respective pre-Amph2 value; ## e p < 0.01, ### e p < 0.001 vs. the corresponding HC group value.

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Fig. 7. Effect of Amph1 on place preference (upper panel) and anticipatory FM 50-kHz USV rate (middle panel) in LC, HC and amphetamine-naive rats (Ctrl) during the CPP test, and on the time course of changes in place preference and anticipatory FM 50-kHz USV rate of HC rats during the CPP test (bottom panel). Place preference was expressed as the difference between the times spent in the amphetamine-paired section of the CPP apparatus during the CPP test and pre-test. CPP data 1-way ANOVA results: F2,23 ¼ 8.07, p ¼ 0.0022; anticipatory FM 50-kHz USV rate data 1-way ANOVA results: F2,21 ¼ 17.6, p < 0.001. * e p < 0.05, ** e p < 0.01, *** e p < 0.001 vs. the respective value for HC rats; the Tukey test.

marked changes in the USV response. Notably, while no sensitization of this response was found in LC rats in the TIPS paradigm, their FM 50-kHz USV responses to Amph9 and Amph10 were significantly stronger than that to Amph1 and showed clearly shorter latency. Hence, it seems that sensitization did develop in at least some LC rats, while at a fairly slow pace, but could have been masked at earlier time points by the tolerance arising over the course of daily drug treatment. One might guess that a longer time between Amph1 and Amph2 would allow a stronger sensitization and a more reliable categorization. Still, in the other studies using long-term amphetamine (Ahrens et al., 2013) or cocaine treatment (Barker et al., in press; Maier et al., 2012), the period of escalating 50-kHz USV response did not exceed one week. Maier et al. (2012) found maximum 50-kHz USV response 7 days after the start of a 3-

week treatment, both in self-administering and in yoked rats, and observed a distinct while transient (of 1 day duration) enhancement of the response after each weekend break in daily drug treatment. This indicates that the possible sensitization developing during the 1st week might have been partially ‘masked’ by tolerance that began to develop over that week. Notably, the number of lever responses rose continuously, suggesting that tolerance affects reward and not the motivational aspect of the progress of drug dependence (Maier et al., 2012). According to the Diagnostic and Statistical Manual of Mental Disorders and the International Statistical Classification of Diseases and Related Health Problems, sensitization is not a key element of drug dependence, and tolerance is a possible, but not necessary one. However, despite the difference in the relative importance of these phenomena clinically, in USV studies much more attention was and still is being given to sensitization. Nonetheless, it is important to study the mechanisms underlying both tolerance and sensitization. We, in contrast to other authors studying sensitization of 50-kHz USV response to psychostimulants, have used an experimental schedule that allows for effective sensitization of the response with but a single dose of amphetamine. The experimental design employed promotes also the emergence of tolerance over the course of the following repetitive drug treatment and finally enables comparing the stability of these characteristics. We did not observe the tolerance previously (Taracha et al., 2012), nor was it seen by Ahrens et al. (2013). This could be due to the rather short period of daily amphetamine treatment in our study (2e3 daily doses before the respective USV session), and to the fact that Ahrens et al. administered the drug to their rats but every 2e3 days. Besides changes in the intensity of FM 50-kHz USV response, the first drug dose caused a reduction in the latency of the response, which greatly differed between HC and LC rats. In the former, the latency showed a major and rapid fall to a permanently low level, whereas in the latter the decrease was slower and reached a similar low level only at the end of the study period. These changes showed no apparent link to the emergence and waning of tolerance of the FM 50-kHz USV response to amphetamine. This observation suggests that the decrease in the latency is not a ‘mechanistic’ consequence of the increased rate of the USV response to the drug, but may be linked to some long-lived or permanent neurobiological change(s) that have little or nothing to do with the rewarding properties of amphetamine. Strangely enough, the repeated drug treatment-related changes in the latency were, so far, not given any attention in all studies on the effect of psychostimulant on the appetitive USV save for our former report (Taracha et al., 2012). 4.3. Repetitive amphetamine treatment effects: LA response Rat LA response to amphetamine is known to undergo sensitization with repeated exposure to the drug (see Kalivas and Stewart, 1991; Robinson and Becker, 1986). In this study, LA response, in contrast to USV response, showed no appreciable difference between LC and HC rats, little or no sensitization, and no signs of tolerance after daily drug treatment (cf. Figs. 3 and 6). Our attempt (not shown) to classify the study rats into low and high responders by their LA responses in the TIPS paradigm (using the mean  2 SEM exclusion criterion similar to that employed for the USV-based categorization) showed no utility of this approach, as the difference faded away over the course of further drug treatment. Besides, at all stages of drug treatment, the LA responses of LC and HC rats showed much smaller inter-individual diversity than the respective USV responses. A dissociation between LA and USV responses to addictive drugs was also reported by others (Ahrens et al., 2013; Maier et al., 2012; Simola et al., 2014). These findings suggest that an enhanced LA response to amphetamine depicts a different

Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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aspect of the progress toward drug dependence than that reflected by changes in FM 50-kHz USV. The USV is mostly dependent on the activity of the brain circuits involving the mesocorticolimbic dopamine system and nucleus accumbens shell (Brudzynski et al., 2011; Burgdorf et al., 2001, 2007; Thompson et al., 2006). However, while the nucleus accumbens is an important neural substrate also in mediating LA responses to psychostimulants (Everitt and Robbins, 2005; Vezina, 2004), these responses are closer related to the function of nucleus accumbens core (Di Chiara, 2002; Li et al., 2004; Parkinson et al., 1999; Sellings and Clarke, 2006) and are greatly affected by the activity of the nigrostriatal dopamine system that supposedly functions as an effector mechanism for the nucleus accumbens (Beeler et al., 2009). 4.4. Drug context-related effects: “anticipatory” FM 50-kHz USVs Fifty-kHz USV is well known to be stimulated by addictive drugrelated cues/context. It can be induced with either passive exposure (Ahrens et al., 2013; Hamed et al., 2012; Taracha et al., 2012; Wright et al., 2012) or self-administration (Browning et al., 2011; Maier et al., 2010, 2012) of addictive substances. In this study, the anticipatory FM 50-kHz USV rate was higher in HC than in LC rats, which is consistent with other reports (Ahrens et al., 2013; Burgdorf et al., 2007; Taracha et al., 2012). The changes in anticipatory USV paralleled those in FM 50-kHz USV responses to repeated amphetamine doses. The high pre-Amph2 FM 50-kHz USV rate in HC rats evidenced their effective conditioning with Amph1. The cause of the reduction in the context-related USV in these rats at the end of daily drug treatment is, however, not clear. It is possible that the relatively short between-dose intervals prevented the emergence of craving or produced a tolerance to the rewarding action of the drug. However, the drug withdrawal-related change in the anticipatory USV demonstrated that the (neuro)biological modifications underlying the depression of the anticipatory USV are relatively short-lived. The latency of FM 50-kHz USV response to amphetamine context was, similarly to that of FM 50-kHz USV response to the drug, significantly lower in HC than in LC rats, but it showed no significant change in either group over the study period. This suggests that these responses have different neurobiological bases. More support for this idea is in microdialysis studies that showed clear distinctions in the nature of accumbal dopaminergic activity and its link to behavior during acquisition and expression of CPP (Weitemier and Murphy, 2009). 4.5. Drug context-related effects: CPP Conditioning is the key factor both in CPP and in the TIPS paradigm. We intended to use CPP both to verify rat categorization based on TIPS-related sensitization of FM 50-kHz USV response to amphetamine and to relate the anticipatory FM 50-kHz USV to the results of CPP test. Hence, Amph1 and Amph2 were given to the subject rats in the CPP apparatus, and CPP training relied on administration of only one vehicle dose and one drug dose. HC rats showed markedly stronger CPP than LC rats. Similar relationship was found using the categorization based on 50-kHz USV response to the first iv dose of amphetamine (Ahrens et al., 2013) or to intracerebral administration of the synthetic m-opioid receptor agonist DAMGO (Burgdorf et al., 2007). The correspondence between categorizations using the various drugs and different routes of administration indicates that FM 50-kHz USV may be useful for identifying rats that differ in their susceptibility to drug dependence also in regard to other addictive drugs. Nonselected rats were reported to vocalize more in the amphetamine-paired (Knutson et al., 1999) or cocaine-paired

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(Meyer et al., 2012) than in the vehicle-paired compartment in CPP tests, which suggests a close relationship between CPP and anticipatory USV. We did not record USVs separately in the two sections of the CPP apparatus, but a confrontation of the respective LA recordings and sonograms showed no association between the USV rate and the rat’s location in either LC or HC rats. While this is in apparent contrast with the reports quoted above, similar observations were reported by Wright et al. (2012) who used morphine in nonselected rats, and by Ahrens et al. (2013) who categorized rats by their 50-kHz USV response to the first amphetamine dose and postulated that the two drug training sessions they used were sufficient for CPP, but not for contextconditioned USV. However, Wright et al. (2012) concluded that CPP and context-conditioned 50-kHz calling “are not generally interchangeable” and offered several explanations for this fact, including a doubt about the relation between 50-kHz USV and reward, an interference from other drug effects, and the possibility that these tests reflect different facets of the reward phenomenon. Trying to explain the relationship between the results of the two tests we took a different approach than that employed in other studies. Namely, we looked at the time course of changes in HC rats’ LA during the CPP test, as only these rats showed tangible changes in this test. One may guess that their initial high rate of FM 50-kHz USV reflected the positive affective state evoked by the drug context, but e for the futile awaiting of the next drug dose e the positive state and the related USV gradually faded away. Hence, the rats began to actively seek the drug, which translated into their spending more time in the drug-paired section. Our data do not support the suggestion of Ahrens et al. that anticipatory USV conditioning needs more training sessions than CPP. Instead, we suppose that anticipatory FM 50-kHz USV and CPP reflect two aspects of the reaction to drug context, namely, the anticipation of and the search for reward, respectively. Thus, the USV-derived information may supplement that stemmed from CPP studies. Further support for this idea is in the lack of correlation between the overall rate of rats’ FM 50-kHz USV during the CPP test and the time spent in the amphetamine-paired compartment. A lack of correspondence between 50-kHz USV emission and rewarding behavior was also reported in adolescent male rats, during both spontaneous and acute opioid- or psychostimulant-modulated social interactions (Manduca et al., 2014). 4.6. Drug context-related effects: LA Neither HC nor LC rats showed a significant change in LA response to the drug context in this study. In contrast, Ahrens et al. (2013) reported an increased LA activity in “high caller” rats (categorized by their USV response to the first drug exposure) anticipating their next amphetamine dose. However, this was only during the 30 s periods immediately preceding next drug doses that were cued with strong stimulus light. Moreover, those rats were kept on a reverse light/dark cycle, with all testing confined to the dark phase. This suggests that anticipatory LA response may need a stronger cue than the drug context used in our study. 5. Concluding remarks As reflected by dynamic changes in rat FM 50-kHz USV responses to amphetamine and amphetamine-paired context, the tested drug regimen effectively induces sensitization and tolerance, and allows assessing the stability of these effects. We hoped to be able, due to the analysis of both USV responses (to amphetamine and drug context) and the better understood LA and CPP reactions, to gain a better insight into the meaning of the former. However, we got no clear-cut idea about the relationship between the results of

Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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Fig. 8. Schematic representation of the findings of this study.

these tests. We can only state that USV, LA and CPP reflect different aspects of the addictive action of amphetamine, and the information derived from these methodologies complement and not duplicate one another. Categorization of male SpragueeDawley rats based on the sensitization of their FM 50-kHz USV response to amphetamine, which was in agreement with the results of CPP test, is relevant to the diversified individual vulnerability of the rats to the addictive properties of this drug, whereas ambulatory response to amphetamine has no such value. This may be related to the fact that whereas CPP and FM 50-kHz USV are under dominant scrutiny of mesocorticolimbic circuits (e.g. see Brudzynski et al., 2011; Burgdorf et al., 2007) involving nucleus accumbens shell, LA control is executed with a significant contribution from the nigrostriatal dopamine system (Beeler et al., 2009) and nucleus accumbens core. A schematic representation of the findings of this study is shown in Fig. 8. Acknowledgments The authors thank Ms. A. Biegaj of the Department of Neurochemistry, Institute of Psychiatry and Neurology, for her excellent technical assistance. The study was supported by the Institute of Psychiatry and Neurology statutory fund no. 501-003-130 and by the National Science Centre of Poland grant no. UMO-2011/03/B/ NZ4/02385. References Ahrens, A.M., Ma, S.T., Maier, E.Y., Duvauchelle, L., Schallert, T., 2009. Repeated intravenous amphetamine exposure: rapid and persistent sensitization of 50kHz ultrasonic calls in rats. Behav. Brain Res. 197, 205e209 http://dx.doi.org/ 10.1016/j.bbr.2010.04.001. Ahrens, A.M., Nobile, C.W., Page, L.E., Maier, E.Y., Duvauchelle, C.L., Schallert, T., 2013. Individual differences in the conditioned and unconditioned rat 50-kHz ultrasonic vocalizations elicited by repeated amphetamine exposure. Psychopharmacology (Berl) 229, 687e700. http://dx.doi.org/10.1007/s00213-0133130-9. Barker, D.J., Bercovicz, D., Servilio, L.C., Simmons, S.J., Ma, S., Root, D.H., Pawlak, A.P., West, M.O., 2013. Rat ultrasonic vocalizations demonstrate that the motivation to contextually reinstate cocaine-seeking behavior does not necessarily involve a hedonic response. Addict. Biol. 18 http://dx.doi.org/10.1111/adb.12044 (in press). Beeler, J.A., Cao, Z.F., Kheirbek, M.A., Zhuang, X., 2009. Loss of cocaine locomotor response in Pitx3-deficient mice lacking a nigrostriatal pathway. Neuropsychopharmacology 34, 1149e1161. http://dx.doi.org/10.1038/npp.2008.117. Browning, J.R., Browning, D.A., Maxwell, A.O., Dong, Y., Jansen, H.T., Panksepp, J., Sorg, B.A., 2011. Positive affective vocalizations during cocaine and sucrose selfadministration: a model for spontaneous drug desire in rats. Neuropharmacology 61, 268e275. http://dx.doi.org/10.1016/j.neuropharm.2011.04.012.

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Please cite this article in press as: Taracha, E., et al., Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment, Neuropharmacology (2014), http://dx.doi.org/10.1016/ j.neuropharm.2014.04.008

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